Structure-Activity Relationships and Physiological Aspects
of New Photosynthetic Electron Transport Inhibitors,
3-Alkylaminoalkyliden-2//-pyran-2,4(3//)-diones (APs)
Tadao Asami, Hiroyuki Koike*, Yorinao Inoue*,
Nobutaka Takahashi, and Shigeo Yoshida
C h e m i c a l R e g u l a t i o n o f Biomechanisms Laboratory and * Solar Energy Research G r o u p ,
T h e I n s t i t u t e o f Physical and Chemical Research 2-1 Hirosawa, W a k o , Saitama 351-01, Japan
Z . N a t u r f o r s c h . 4 3 c , 8 5 7 - 8 6 1 (1988); received July 22, 1988
C o n j u g a t e d E n a m i n e , Photosynthetic Electron Transport I n h i b i t o r , 3 - A l k y l a m i n o a l k y l i d e n - 2 / / p y r a n - 2 , 4 ( 3 / / ) - d i o n e s , I n h i b i t o r B i n d i n g Site of D 1 Protein, Thermoluminescence
3 - A m i n o a l k y l i d e n e - 2 / / - p y r a n - 2 , 4 ( 3 / / ) - d i o n e s ( A P s ) , possessing a conjugated enamino moiety
w h i c h is c o m m o n t o cyanoacrylates and 2-aminoalkylidenecyclohexane-l,3-diones ( A C s ) , were
f o u n d as a new class of photosynthetic electron transport inhibitors. A l t h o u g h the structural
r e q u i r e m e n t o f A P s for photosynthetic electron transport i n h i b i t i o n was very similar to that of
cyanoacrylates and A C s , thermoluminescence measurements indicated that the b i n d i n g manner
o f A P s t o D 1 p r o t e i n was totally different f r o m that of other inhibitors.
Introduction
There are many chemical classes of photosynthetic
electron transport inhibitor herbicides, and some of
them have played a great role in physiological investigation on the photosynthetic electron transport system. Designing of new type of such inhibitors which
block the electron flow at a specific site, is still a very
hard task, although the researches on the classical
inhibitors have revealed their binding modes and/or
site(s) at the //ííer-protein of chloroplast level. The
behavior of the inhibitors at the intra-protein level
and/or the actual form of the binding niche have been
retained as very curious problems. In order to elucidate the interaction between those type inhibitors
and their binding sites, various new approaches will
come in the next decade, e.g. topological analyses for
a dynamic state of receptor (binding) protein, modification of peptide unit(s) by gene technology and
computer simulation to optimize the effect of inhibitors. Discovery of new classes of photosynthetic
inhibitors and subsequent activity-structure studies
may support to accumulate more information for
such new approaches.
Abbreviations:
AP(s),
3-aminoalkylidene-2//-pyran2 , 4 ( 3 / / ) - d i o n e ( s ) ; A C ( s ) , 2-aminoalkylidenecyclohexanel , 3 - d i o n e ( s ) ; b r o m o p y r i d i n o l , 2-(a-bromooctyl)-3,5-dibrom o - 2 - m e t h y l p y r i d i n e - 4 - o l ; PS, photosystem; T L , thermoluminescence.
R e p r i n t requests to D r . S. Yoshida.
Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen
0341 - 0382/88/1100- 0806 $01.30/0
Cyanoacrylates (1) [1, 2] and aminoalkylidenecyclohexane-diones (ACs) (2) [3] are new type of
potent inhibitors for the photosynthetic electron
transport. Both involves a conjugated enamine system as an essential functionality for their activity [4],
Furthermore, the character of substituents around
the conjugated enamine moiety of those two types
show similar trends in lipophilicity to reach the
optimum activity, namely they need the suitable disparity of lipophilic and relatively hydrophilic functionalities. These facts indicates that both chemical
classes might have a similar binding niche on D 1
protein of the PS II judging from the structural similarity.
According to the structural relation to A C series,
other six membered ring compounds carrying a 1,3dicarbonyl-2-aminoalkylidene system were examined
with the photosynthetic electron transport inhibition
assay. Among them 3-alkylaminoalkylidene-2//pyran-2,4(3//)-dione (AP) derivatives (3) were
found as potent photosynthetic electron transport inhibitors [4], In this paper we describe the effects of
lipophilic substituents around the conjugated
enamine part of A P molecules and the binding site of
this inhibitor series analyzed by the thermoluminescence band measurements.
Materials and Methods
The structures of synthetic chemicals were confirmed by various 'H NMR spectra which were
recorded on instrumental analyses: 'H NMR (JEOL
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T. Asami et al. • Inhibition of PS II by 3-Alkylaminoalkylidenepyran-2.4-diones
858
MH-100 and FX-100 spectrometers, 100 MHz),
13
C N M R spectra (FX-100, 25.5 MHz), IR spectra
( J A S C O A-400) and U V spectra ( H I T A C H I
M O D E L 200-20).
In this study, 3-acyl-6-methyl-2//-pyran-2,4(3//)dione and 6-methyl-2//-pyran-2,4(37/)-dione were
used as starting materials for synthetic procedures
described in the previous paper [4],
Table I. Photosynthetic electron transport i n h i b i t i o n of
various APs.
Hill reaction assay
Compounds
R1
R2
p/50
Spinach thylakoids were prepared as described in
[5]. Thylakoids (final concentration 0.5 pg Chl/ml)
were suspended in 50 mM H E P E S - N a O H (pH 7.0),
10 mM NaCl, 20 mM methylamine, 50 pM D C I P , and
inhibitors if added. Electron transport activity was
measured by monitoring D C I P photoreduction at
600 nm with Shimadzu UV-300 spectrophotometer.
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
DCMU
— (CH-, . O C T L C H ,
- ( C H 2 5CH3
- ( C H 2 9CH3
- ( C H 2 13CH3
- ( C H 2 5CH3
— ( C I L 7CH3
- ( C H 2 9CH3
- ( C H 2 11CH3
- ( C H 2 .3CH3
~ ( C H 2 5CH3
- ( C H 2 7CH3
— (CTL 9CH3
- ( C H 2 11CH3
- ( C H 2 13CH3
- ( C H 2 5CH3
— (CH> 9CH3
— (CTL 13CH3
- ( C H 2 3CH3
- ( C H 2 5CH3
— (CH-, 9CH3
" ( C H 2 13CH3
- H
- H
- H
- H
-CH3
-CH,
-CH,
-CTL
-CH3
-CTLCTL
-CH.CH,
-CH,CH,
-CH.CH,
-CH.CTL
~(CH2)2CH3
-(CH2)2CH3
-(CH2)2CH3
-(CH2)2CH3
-(CH2)2CH3
-(CH2)2CH3
-(CH2)2CH3
3.5 >
3.5 >
3.5>
3.5>
5.6
5.8
6.4
7.3
7.2
5.4
6.6
7.4
7.5
7.1
5.0
6.2
5.0
3.5>
3.5>
3.8
3.5>
7.3
Thermoluminescence
measurement
Thylakoids were diluted (0.25 mg Chl/ml) with
25% (v/v) glycerol, 10 mg MgCl 2 , and 50 mM
H E P E S - N a O H (pH 7.0), and were illuminated with
orange light for 45 sec then dark adapted for 5 min at
room temperature. Thermoluminescence was measured as described in [6]. The samples were illuminated with xenon flashes, rapidly cooled to —196 °C,
then heated at a rate of 0.8 °C/sec.
Results and Discussion
Structure-activity
8
relationships
The chemical modification of the TV-substituent
(R 1 ) and the alkylidene part (R 2 ) in A P series were
carried out and the bioassay results are listed in
Table I. When R 2 is fixed to —CH 3 or —C2H5 groups,
a proper range of lipophilicity in R 1 caused very high
inhibition, however, only negligible activity was observed in the case of — H or —C4H9 groups for R 2 .
Thus, the suitable combination of R 1 and R 2 is necessary for A P to show the inhibition on the photosynthetic electron flow, as well as the case of cyanoacrylates and A C . The lipophilic effect of R 1 was
demonstrated by the change of the alkyl chain length
in the A P series which carries a ethyl group as R :
(Fig. 1). The effect was optimized with the dodecyl
group and the rather wide range (from C 8 to C 14 or
more) of lipophilicity was shown to be effective for
the high level (more than 6.0 in p/ 50 values) of
activity.
o
IO
CL
7
/
_
6"
5
4
6
8
10
12
14
16
n
Fig. 1. Diagrammatic representation of the change o f p/ s o
f o r A P s of increasing alkyl chain length in R 1 .
On the contrary, R 2 of A P was critically limited to
the narrow range (between Q and C 3 ) of the
lipophilicity as illustrated in Fig. 2. Since this pecul-
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859 T. Asami et al. • Inhibition of PS II by 3-Alkylaminoalkylidenepyran-2.4-diones
LO
the further investigation of binding studies. For the
analysis on the binding mode of D 1 protein inhibitors, the thermoluminescence measurement [9] is
one of the most effective to make evidences at the
intra-protein level.
CL
Thermoluminescence
Fig. 2. Diagrammatic representation o f the change of p/ 5 0
f o r A P s of increasing a l k y l chain length in R : .
iar nature of R 2 in A P is similar to that in cyanoacrylates [7, 8], it may be possible to think about a common domain for the enamine functions of both types
in the D 1 protein.
Consequently the combination of dodecyl for R 1
and ethyl for R 2 has been recognized as the optimum
distribution for the lipophilicity around the enamine
functionality for A P series to inhibit the photosynthetic electron flow. In this case the optimized
inhibition of AP (16) was surpassing the effect of
DCMU.
It is noteworthy that APs (5—7) with the methylidene structure (R 2 = —H) was totally inactive. This
point is one of the most significant structural difference between APs and ACs which exhibited very
high activity with the methylidene structure [3]. Furthermore another basic difference between those two
series was recognized in the location of lipophilic
moieties in their molecules. This means that a
lipophilic N-substituent is necessary for the active
APs, while the active ACs carry a polar group as the
corresponding part. Thus, a curious problem concerning the binding site(s) of those new inhibitors
occurred again from the structural consideration, although their modes of action had not been examined
in detail.
As described above, the vicinity of the conjugated
enamine system of APs, ACs and cyanoacrylates
should be a key part to generate the photosynthetic
electron transport inhibition. Consequently an accurate evaluation of their inhibition manners depending on the enamine structures should be required for
method
A number of inhibitors which interrupt the electron flow at photosystem II (PS II) has been reported and the mode of action has been studied extensively. These PS II inhibitors were classified into
two major groups, an urea/triazine type and a phenol
type by their chemical structures and inhibitory patterns [10]. Both types of inhibitors inhibit the electron flow between Q A and O b by binding at the plastoquinone (QB) domain resulting in replacement of
the quinone with inhibitors. Since an inhibitor is replaceable with another inhibitor of different class,
the presence of a common binding domain on PS II
is suggested in the receptor of those inhibitors. There
is a clear correlation between the binding sites and
the structure of these two groups. The urea/triazine
type inhibitors have common structural features of
lipophilic part(s) in association with the azo-functionalities [ - C ( = X ) - N < ] [11]. Although ureas and
triazines were classified into one group of D 1 protein
inhibitors, significant gaps of activity were often observed by the use of triazine-resistant D 1 proteins
which were obtained from mutant plants [12].
The redox state of Q A and Q B in PS II can be
monitored by thermoluminescence (TL) measurements, and the TL glow curve shows a main band at
around 30 °C (B-band) which is attributed to recombination between S2 (and/or S3) and QB . Inhibitors
cause a lower temperature shift of the TL-band due
to recombination between S2 and QÄ (Q-band)
[13—15]. The peak temperature of the Q-band is dependent on the treatment of a particular inhibitor
class [9]. From the peak emission temperature of Qband, the orthodox inhibitors are classified into three
groups; ureas, triazines and phenols in agreement
with differences indicated by the mutant experiments
[13]. Thus, the TL glow curve measurement is a
powerful tool for grouping new inhibitors.
Table II shows the TL glow peaks under the presence of various inhibitors. The peak temperature of
Q-band in the presence of DCMU, atrazine and
ioxynil were + 7 , + 1 , and —7 °C, respectively. The
peaks of TL glow curves by A C ( + 8 °C) and cyano-
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860
T. Asami et al. • Inhibition of PS II by 3-Alkylaminoalkylidenepyran-2.4-diones
Table I I . Thermoluminescence glow peaks under the presence of various inhibitors.
Compounds
Concentration [PM]
F m a x [°C]
DCMU
Propanil
Bromacil
Buthidazole
Atrazine
Ioxynil
Bromopyridinol*
Cyanoacrylate
AC
AP
Control
10
10
10
10
6
100
10
10
10
10
+ 7
+ 6
+ 8
+ 6
+ 1
-7
-6
+ 6
+ 8
+ 18
+ 38
—
Control
DCMU (10 pM)
AP (15 p M)
A b b r e v i a t e d name o f 2-(a-bromooctyl)-3,5-dibromo-2m e t h y l p y r i d i n e - 4 - o l ; see ref. [16].
acrylates ( + 6 ° C ) were very similar to that of
D C M U , therefore those new inhibitors should be
classified into the DCMU-type inhibitor. On the
other hand, the glow peak of A P ( + 2 0 °C) was
found to be intermediate between B-band and
DCMU-induced Q-band. There have been no report
on the inhibitor which shows the emission peak at
such a high temperature. This suggests that AP binds
to the D 1 protein with a different manner from those
of other inhibitors.
Another characteristic effect of AP in the TL
measurement was observed in the band height after
the second flash, which was higher than that of the
first flash whereas the other types of inhibitors gave a
constant band height for every flash (Fig. 3). The
height of the B-band which arises from the recombination between S2/S3 and QB in the absence of inhibitors shows a quadruple oscillation with a maximum at 2nd flash and a minimum at 4th flash. It
should be noted that the concentration of the inhibitors (15 pM) tested in Fig. 3 was sufficient for
complete inhibition of electron flow (see also Table
I). Thus, the anomalous behavior of AP in the TL
measurement is not due to insufficient blocking of
the electron flow.
The oscillation of Q-band induced by atrazine, AC
and ioxynil showed practically the same pattern as
that of D C M U although the emission temperature is
different with each other. The difference in the peak
temperature might reflect the interaction between
QA and inhibitors. Thus, as far as the results of TL
measurements, APs should be classified as totally
Fig. 3. Oscillation patterns of the intensity of thermoluminescence w i t h flash times under the n o r m a l condition
and under the presence o f D C M U and A P .
new type inhibitors in the binding manner at the D 1
protein.
Conclusion
Our present study indicated that the compounds
holding a conjugated enamine system should be an
effective functionality to inhibit the photosynthetic
electron transport. In spite of the similarity in the
chemical nature of the enamine systems, there are
obvious differences in their inhibition mode due to
variation of the substitutional pattern around the system. This fact leads us to the assumption that a conjugated enamine system presumably has a potent affinity to peptide bond(s) and/or residue(s) of amino
acid(s) in the D I protein. According to this idea,
those inhibitors described in this paper might be useful as good probes to investigate a niche of herbicides
in the D 1 protein because of easiness in their structural modification.
Further characterization for the unique binding
site of AP is now under way using various TL techniques and mutant chloroplasts.
Acknowledgements
A part of this research was supported by Nissan
Science Foundation. The authors gratefully acknowledge Dr. J. N. Phillips (CSIRO, Australia) for his
kind supply of cyanoacrylates.
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861 T. Asami et al. • Inhibition of PS II by 3-Alkylaminoalkylidenepyran-2.4-diones
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